Etiologic Risk Group (see link below):Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia
*Background/Disease Information (sort of like the Intro to your Mini Research Write up): Epidemic typhus is caused by Rickettsia prowazekii bacteria and is spread to humans through lice or ticks. In the US, the bacterium is most commonly found on the flying squirrel. Contraction of typhus can be prevented through a series of vaccines. It is uncommon in the US, but a few cases of epidemic typhus have been reported in Austin, Texas. Typhus is more common in areas of the world that have low hygiene and cold temperatures. Once the disease has been contracted, it can become dormant and then re-activate. This is known as Brill-Zinsser disease and is more common in the elderly. If left untreated, the disease has the potential to be fatal. Death occurs in 10-60% of patients. Symptoms include chills, confusion, stupor, rash, high fever, cough, headache, arthralgia, low blood pressure, severe headache and muscle pain. A CBC (complete blood count) can be used to diagnose the disease and shows levels of typhus antibodies, levels albumin, sodium level, and liver enzyme levels. Common treatments include antibiotics such as tetracycline. Prevention measures include bathing, avoiding areas that are infested with rat fleas or lice, boiling infected clothing for a period of 5 days, and using insecticides.
Enzyme Assay information: (3R)-3-hrdroxyacyl-[acyl-carrier protein] plus NADP+; spectrophotometric
Substrate Info (Dr. B') - 100113 May also dilute in Tris - talk to Kaarthik.
NADPH - sigma N6505 http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/2/n6505pis.Par.0001.File.tmp/n6505pis.pdf Preparation Instructions b-NADPH is soluble in 0.01 M sodium hydroxide (50 mg/ml), yielding a clear, light yellow solution. Storage/Stability It is recommended to store Products N1630, N7505, and N6505 desiccated at –20 °C protected from light. Product N9910 can be stored at room temperature. The normal impurities and/or decomposition products are b-NADP and monophosphoadenosine 5¢- diphosphoribose. It is recommended to prepare solutions fresh and use promptly, unless you are sure this is an unnecessary precaution for your work. However, it has been reported that a 0.5 mM solution in 0.02 M NaOH (pH 12.3) showed no loss of purity in a week at 4 °C or -85 °C, but a 13% loss at –20 °C.3
For NADH and NADPH - the concern is that these are reduced and don't want them to be oxidized. So, want to reduce their exposure to oxygen - hence the NaOH.
Structure Available (PDB or Homology model) PDB ID: 3F9I
Current Inhibitors: Flavonoids, EGCG in green tea
Expression Information (has it been expressed in bacterial cells):Expressed in E. coli cells
Purification Method:Nickel column + FPLC
Image of protein:
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MIDLTGKTSLITGASSGIGSAIARLLHKLGSKVIISGSNEEKLKSLGNALKDNYTIEVCN LANKEECSNLISKTSNLDILVCNAGITSDTLAIRMKDQDFDKVIDINLKANFILNREAIK KMIQKRYGRIINISSIVGIAGNPGQANYCASKAGLIGMTKSLSYEVATRGITVNAVAPGF IKSDMTDKLNEKQREAIVQKIPLGTYGIPEDVAYAVAFLASNNASYITGQTLHVNGGMLM VX
Length of your protein in Amino Acids: 242
Molecular Weight in kiloDaltons: 25870.1 kD
Molar Extinction coefficient of your protein at 280 nm wavelength:10680 M-1 cm-1, 10430 M-1 cm-1
*Target (protein/gene name): Fab G 3-ketoacyl-(acyl-carrier-protein) reductase
*NCBI Gene # or RefSeq#: 883564
*Protein ID (NP or XP #) or Wolbachia#: NP_221114.1
*Organism (including strain): Rickettsia prowazekii
Etiologic Risk Group (see link below): Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Epidemic typhus is caused by Rickettsia prowazekii bacteria and is spread to humans through lice or ticks. In the US, the bacterium is most commonly found on the flying squirrel. Contraction of typhus can be prevented through a series of vaccines. It is uncommon in the US, but a few cases of epidemic typhus have been reported in Austin, Texas. Typhus is more common in areas of the world that have low hygiene and cold temperatures. Once the disease has been contracted, it can become dormant and then re-activate. This is known as Brill-Zinsser disease and is more common in the elderly. If left untreated, the disease has the potential to be fatal. Death occurs in 10-60% of patients. Symptoms include chills, confusion, stupor, rash, high fever, cough, headache, arthralgia, low blood pressure, severe headache and muscle pain. A CBC (complete blood count) can be used to diagnose the disease and shows levels of typhus antibodies, levels albumin, sodium level, and liver enzyme levels. Common treatments include antibiotics such as tetracycline. Prevention measures include bathing, avoiding areas that are infested with rat fleas or lice, boiling infected clothing for a period of 5 days, and using insecticides.
Link to TDR Targets page (if present): Not present
Link to Gene Database page: http://www.ncbi.nlm.nih.gov/gene/883564
Essentiality of this protein: Very essential in the biosynthesis of fatty acids pathway
Complex of proteins?: Tetramer in solution
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
*EC#: 1.1.1.100
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.100
Enzyme Assay information:
(3R)-3-hrdroxyacyl-[acyl-carrier protein] plus NADP+; spectrophotometric
Substrate Info (Dr. B') - 100113
May also dilute in Tris - talk to Kaarthik.
NADPH - sigma N6505
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/2/n6505pis.Par.0001.File.tmp/n6505pis.pdf
Preparation Instructions
b-NADPH is soluble in 0.01 M sodium hydroxide
(50 mg/ml), yielding a clear, light yellow solution.
Storage/Stability
It is recommended to store Products N1630, N7505,
and N6505 desiccated at –20 °C protected from light.
Product N9910 can be stored at room temperature.
The normal impurities and/or decomposition products
are b-NADP and monophosphoadenosine 5¢-
diphosphoribose.
It is recommended to prepare solutions fresh and use
promptly, unless you are sure this is an unnecessary
precaution for your work. However, it has been
reported that a 0.5 mM solution in 0.02 M NaOH
(pH 12.3) showed no loss of purity in a week at 4 °C or
-85 °C, but a 13% loss at –20 °C.3
For NADH and NADPH - the concern is that these are reduced and don't want them to be oxidized. So, want to reduce their exposure to oxygen - hence the NaOH.
Structure Available (PDB or Homology model)
PDB ID: 3F9I
Current Inhibitors: Flavonoids, EGCG in green tea
Expression Information (has it been expressed in bacterial cells): Expressed in E. coli cells
Purification Method: Nickel column + FPLC
Image of protein:
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MIDLTGKTSLITGASSGIGSAIARLLHKLGSKVIISGSNEEKLKSLGNALKDNYTIEVCN
LANKEECSNLISKTSNLDILVCNAGITSDTLAIRMKDQDFDKVIDINLKANFILNREAIK
KMIQKRYGRIINISSIVGIAGNPGQANYCASKAGLIGMTKSLSYEVATRGITVNAVAPGF
IKSDMTDKLNEKQREAIVQKIPLGTYGIPEDVAYAVAFLASNNASYITGQTLHVNGGMLM
VX
Length of your protein in Amino Acids: 242
Molecular Weight in kiloDaltons: 25870.1 kD
Molar Extinction coefficient of your protein at 280 nm wavelength: 10680 M-1 cm-1, 10430 M-1 cm-1
TMpred graph Image:
*CDS Gene Sequence (paste as text only):
CAAACAATATTTAAAAGAACATCCACAAATTTCCAACGAAATTGAGAAAATAATACGAGAAAAATCGTCA
GCAATTACTAATATAAATCTTGATCAAACGGAGGAATAAAATGATTGATTTAACAGGTAAAACTTCCTTA
ATTACAGGTGCTTCAAGTGGCATAGGAAGTGCCATAGCGCGGTTACTACATAAACTCGGGAGCAAGGTAA
TTATTAGCGGTAGTAATGAAGAAAAATTAAAATCACTTGGCAATGCTTTAAAAGATAATTATACCATAGA
AGTATGTAATCTTGCGAATAAGGAAGAATGTAGTAATTTAATATCTAAAACATCAAATTTAGATATTTTA
GTATGTAATGCCGGTATTACTAGTGATACACTCGCAATTAGAATGAAAGATCAAGATTTCGACAAAGTTA
TTGATATTAATTTGAAAGCCAATTTTATTTTAAATCGTGAAGCAATAAAAAAAATGATACAAAAAAGGTA
TGGACGCATTATTAACATATCTTCAATAGTAGGAATTGCAGGTAACCCAGGACAGGCTAATTATTGCGCT
TCTAAAGCAGGTTTAATAGGTATGACCAAATCGCTATCTTACGAAGTTGCAACTAGAGGGATTACGGTTA
ATGCAGTAGCACCAGGATTTATTAAATCAGATATGACTGATAAACTCAATGAAAAACAACGAGAAGCAAT
AGTACAAAAAATTCCACTAGGTACTTATGGTATACCAGAAGACGTTGCATACGCAGTCGCATTTTTAGCA
AGCAATAATGCATCATATATTACTGGTCAAACTCTACATGTAAACGGTGGTATGTTAATGGTATAAAATT
TTATGCTAGCAAAAAATAATTTTTTGTGCTAGAAGTTCTTTTAAAGAGAAAATGCTTATGAATATTTATT
TCAAATATTCATAGCAATGAAAATAAAATCTAAT
*GC% Content for gene: 30.6%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATGATTGACCTCACGGGCAAAACTTCTCTGATCACGGGTGCCAGCTCTGGTATCGGTTCT
GCAATCGCGCGTCTGCTGCACAAACTGGGCTCTAAAGTTATTATCTCTGGTTCTAACGAA
GAAAAACTGAAGTCTCTCGGTAACGCGCTCAAAGACAACTACACCATCGAAGTTTGTAAC
CTCGCGAACAAAGAAGAATGCAGCAATCTGATCTCTAAAACCTCTAATCTGGACATCCTG
GTTTGCAATGCGGGCATCACCTCTGACACCCTGGCGATCCGTATGAAAGACCAGGACTTT
GACAAAGTCATCGATATCAACCTGAAAGCGAACTTCATCCTGAACCGTGAAGCGATCAAG
AAGATGATCCAGAAACGTTACGGTCGTATTATCAACATCTCCTCTATCGTTGGTATTGCG
GGTAACCCAGGTCAGGCGAACTACTGCGCATCTAAAGCGGGCCTGATCGGCATGACCAAA
TCTCTGTCTTACGAAGTTGCGACCCGCGGTATCACCGTCAACGCCGTTGCGCCAGGTTTC
ATCAAATCTGACATGACTGACAAGCTGAATGAAAAACAGCGTGAGGCAATCGTTCAGAAA
ATCCCGCTGGGTACCTACGGTATTCCAGAAGACGTGGCTTACGCGGTTGCGTTCCTGGCG
TCTAACAACGCGTCTTACATTACCGGTCAAACCCTGCATGTTAACGGTGGTATGCTGATG
GTTTAA
*GC% Content for gene (codon optimized): 48.6%
Primer design results for pNIC-Bsa4 cloning:
1 ATGATTGACCTCACGGGCAAA 21
2 GCAGAACCGATACCAGAGCTGGCACCCGTGATCAGAGAAGTTTTGCCCGTGAGGTCAATC 60
3 GCTCTGGTATCGGTTCTGCAATCGCGCGTCTGCTGCACAAACTGGGCTCTAAAGTTATTA 60
4 GAGAGACTTCAGTTTTTCTTCGTTAGAACCAGAGATAATAACTTTAGAGCCCAGTTTGTG 60
5 TAACGAAGAAAAACTGAAGTCTCTCGGTAACGCGCTCAAAGACAACTACACCATCGAAGT 60
6 ATCAGATTGCTGCATTCTTCTTTGTTCGCGAGGTTACAAACTTCGATGGTGTAGTTGTCT 60
7 AAAGAAGAATGCAGCAATCTGATCTCTAAAACCTCTAATCTGGACATCCTGGTTTGCAAT 60
8 CTTTCATACGGATCGCCAGGGTGTCAGAGGTGATGCCCGCATTGCAAACCAGGATGTCCA 60
9 CCTGGCGATCCGTATGAAAGACCAGGACTTTGACAAAGTCATCGATATCAACCTGAAAGC 60
10 CATCTTCTTGATCGCTTCACGGTTCAGGATGAAGTTCGCTTTCAGGTTGATATCGATGAC 60
11 CGTGAAGCGATCAAGAAGATGATCCAGAAACGTTACGGTCGTATTATCAACATCTCCTCT 60
12 GTTCGCCTGACCTGGGTTACCCGCAATACCAACGATAGAGGAGATGTTGATAATACGACC 60
13 ACCCAGGTCAGGCGAACTACTGCGCATCTAAAGCGGGCCTGATCGGCATGACCAAATCTC 60
14 CGTTGACGGTGATACCGCGGGTCGCAACTTCGTAAGACAGAGATTTGGTCATGCCGATCA 60
15 GCGGTATCACCGTCAACGCCGTTGCGCCAGGTTTCATCAAATCTGACATGACTGACAAGC 60
16 GATTTTCTGAACGATTGCCTCACGCTGTTTTTCATTCAGCTTGTCAGTCATGTCAGATTT 60
17 TGAGGCAATCGTTCAGAAAATCCCGCTGGGTACCTACGGTATTCCAGAAGACGTGGCTTA 60
18 TGTAAGACGCGTTGTTAGACGCCAGGAACGCAACCGCGTAAGCCACGTCTTCTGGAATAC 60
19 CGTCTAACAACGCGTCTTACATTACCGGTCAAACCCTGCATGTTAACGGTGGTATGCTGA 60
20 TTAAACCATCAGCATACCACCGTTAACAT 29
DNAWorks Output Link:
https://docs.google.com/file/d/0B_Gl3lMyhDsoQ0pkekVLcHZTVVE/edit?usp=sharing
Primer design results for 'tail' primers (this is just 2 sequences):
Forward Primer:
5’ TACTTCCAATCCATGATTGACCTCACGGGCAA 3'
Reverse Primer:
5’ GTGGTATGCTGATGGTTTAACAGTAAAGGTGGATA 3’